Program Director/Principal Investigator (Last, First, Middle): PROJECT SUMMARY/ABSTRACT We propose to study membrane-active proteins and peptides that cause configurational changes in cell membranes, including pore formation and membrane fusion. Pore-forming peptides are animals'(including human's) gene-encoded innate antimicrobials that kill microbes by forming pores in their membranes. Clarification of their molecular mechanisms will facilitate their therapeutic applications. Our studies of this problem have led to a free energy description of peptide-lipid interactions. In specific aim 1 we will extend such studies to the kinetics of pore-formation. Studies of these relatively simple peptides have also contributed to the development of experimental methods that can be used for more complex protein-membrane interactions. Pore-forming proteins include apoptosis regulating proteins, in particular Bax which is soluble in the cytosol under normal conditions, but in the presence of apoptotic stimuli, it translocates to the outer mitochondrial membrane and induces cytochrome c release by forming pores. In specific aim 2, we will analyze the molecular mechanism of pore formation by Bax. The viral fusion protein (hemagglutinin or HIV gp41) inserts the N-terminal fusion peptide into the target membrane to initiate membrane fusion. In specific aim 3 we will study the effect of fusion peptides on the first step of membrane fusion. The molecular mechanisms of membrane-peptide (or -protein) interactions must have a structural basis. Obtaining the structural information for each system is our primary goal. Using oriented membranes containing peptides, we have developed methods for measuring the orientation of the peptides, measuring the membrane thickness as a function of peptide-lipid ratio, detecting and measuring the size of transmembrane pores, and resolving the structures of the peptide-induced pores. We will extend these methods to study pore-forming proteins. The biological functions of membrane-active proteins and peptides will be simulated in kinetic experiments with giant unilamellar vesicles (GUVs). The surface area change and the volume change of the GUV in the kinetic process will be measured. We will then interpret the kinetic results in terms of the structural basis. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page